Berg, A.H.

Olsson, P.-E.

Förlin, L.

Abstract [en]

Environmental oestrogens are natural or synthetic substances present in the environment, which imitate the effects of endogenous oestrogen. Oestrogenic substances were identified by gas chromatography/mass spectrometry in effluent water from a Swedish sewage treatment works receiving mainly domestic wastewater. Substances found include the synthetic oestrogen used in contraceptives 17 alpha-ethinyloestradiol (4.5 ng l(-1)), the natural oestrogens oestrone (5.8 ng l(-1)) and 17 beta-oestradiol (1.1 ng l(-1)), and the weaker non-steroidal oestrogens 4-nonylphenol (840 ng l(-1)) and bisphenol A (490 ng l(-1)). Ethinyloestradiol exceeded levels shown to be oestrogenic to fish by 45 times. The oestrogenicity of the effluent water was investigated by introducing juvenile rainbow trout (Oncorhynchus mykiss) in cages downstream of the sewage treatment works. After 2 weeks, all oestrogens indicated were present in the bile of the fish, and the oestrogen inducible protein, vitellogenin, was found in large amounts in the plasma (1.5 mg ml(-1)), as determined by enzyme-linked immunosorbent assay and Western blotting. Thus, a widely used synthetic oestrogen affects the endocrine systems of fish exposed to sewage effluent water.

Adolfsson-Erici, Margaretha

Abstract [en]

This work explores the usefulness of fish bile analysis in combination with biomarkers for identifying and evaluating new environmental contaminants in the aquatic environment. It illustrates how bile analysis can be used together with biomarkers to assess the causes of estrogenic effects, to identify chemicals in the aquatic environment that are taken up by fish, and to monitor environmental exposure.

In a first application, fish exposed to sewage treatment plant effluent were studied. Elevated levels of vitellogenin in the exposed fish demonstrated that estrogenic effects occurred. Several estrogen disrupting substances were identified in the fish bile, and analysis of water samples confirmed that these substances were present in the effluent. The synthetic estrogen 17a-ethinylestradiol, which is known to be present in sewage treatment plant effluent, was shown for the first time to be taken up by fish. Considering the reported potencies of the detected substances, it was concluded that 17a-ethinylestradiol was the major contributor to the estrogenic effects.

Chemical analysis of bile was used to identify rubber additives that were released from tires immersed in water. The bile of rainbow trout held in the water contained high levels of metabolites of PAHs and aromatic nitrogen compounds. Several biomarkers were also measured in the exposed fish, and EROD induction and oxidative stress were observed. Based on the bile analysis observations together with knowledge of toxicological mechanisms, it was postulated that the EROD induction was due to the PAHs, while aromatic nitrogen compounds caused the oxidative stress.

Resin acids in fish bile proved to be a good indicator of exposure in a chronic long-term study of rainbow trout exposed to effluent from a total chlorine free (TCF) pulp mill. Elevated levels of GST (gluthatione-S-transferase) and GR (gluthatione reductase) activity, and the presence of DNA adducts after a two month recovery period, indicated that compounds in the pulp mill effluents have persistent effects. In addition to characterising the exposure of the fish to the effluent, the analysis of the resin acids in the bile provided evidence of accidents in the pulp mill that the existing process monitoring system had not detected.

Resin acids in bile were also found to be a valuable indicator of exposure to pulp mill effluents for eelpout living in the Baltic Sea. A correlation between resin acid levels in bile and skewed sex ratios provided an important link in the chain of evidence that substances in the pulp mill effluents cause male bias of the eelpout embryos.

A particularly good example of the potential of bile analysis was the identification of a previously unknown environmental contaminant. A large peak was observed in the bile extracts of fish that had been exposed to sewage treatment plant effluent. This peak was identified as triclosan, which demonstrated its presence in sewage treatment plant effluent. Other work went on to show that it is a common contaminant of the aquatic environment. The ability of fish to concentrate contaminant metabolites in bile to levels very much higher than in the environment, and the comparatively low levels of analytic interferences, make bile a particularly attractive matrix to search for new, unknown organic pollutants

Pettersson, Maria

Abstract [en]

The aim of this thesis was to investigate if endocrine disruption related to the release of estrogenic and/or androgenic substances via sewage treatment plant and pulp mill effluents is significant in Swedish waters. Chemical analysis of bile fluid in combination with measurement of biomarkers was used for the evaluation of internal exposure and effects in fish.

Exposure to estrogenic substances was demonstrated by pronounced induction of the yolk protein precursor vitellogenin in fish exposed in cages downstream of a small domestic Swedish sewage treatment plant. Estrogenic substances identified in bile from the fish included the natural estrogens, the synthetic estrogen ethinylestradiol and some estrogenic phenols. The bile levels of estrogenic substances corresponded well with the plasma levels of vitellogenin. Significant concentrations of the estrogenic substances were also determined in the effluent water, and it was suggested that the level of ethinylestradiol was sufficient to explain a major part of the observed estrogenicity. Low or no induction of vitellogenin was observed in fish when exposed to final effluent from two modern Swedish sewage treatment plants, and it was concluded that a well functioning biological treatment reduces the amounts of estrogenic substances from the sewage water and that slow sand filtration contributes to further reduction. A field survey along the Baltic Sea coast revealed no signs of estrogenic or androgenic disruption in recipients of sewage treatment plants or pulp mills. This is likely due to efficient water treatment and high dilution rates in the recipients. Endocrine disruption might still be of local concern in receiving waters of less treated effluents that have low dilution rates.

A sensitive method for determination of estrogenic substances in water was developed, which will make it possible to establish bioconcentration relationships between water and fish, and dose-response relationships with biomarkers in fish.